The present invention relates to peptide chemistry. More particularly, it relates to the synthesis of anti-mitotic compounds that have use as anti-proliferative and anti-cancer agents. The compounds, which are synthetic peptide derivatives, are similar to natural substances, called diazonamides and analogues, originally isolated from a marine invertebrate, Diazona angulata. This invention relates to anti-mitotic diazonamides and analogues thereof, their use as anti-proliferative and anti-cancer agents, and methods of synthesis.
Cell mitosis is a multi-step process that includes cell division and replication. It is characterized by the intracellular movement and segregation of organelles, including mitotic spindles and chromosomes. Organelle movement and segregation are facilitated by tubulin polymerization. Microtubules are polymers of globular tubulin subunits formed into cylindrical tube structures. The dynamic polymerization of these structures is essential for cell mitosis. Gelfand and Bershadsky, “Microtubule dynamics: mechanism, regulation and function,” Ann Rev Cell Biol 1991 7:93-116.
Antimitotic compounds such as colchicine, vinblastine and taxol can inhibit microtubule polymerization. These compounds restrict tubulin polymerization and cells treated with these compounds become arrested in mitosis. During mitosis, tubulin subunits of the mitotic spindle are exchanged in a continual process with the pool of cellular tubulin. Taxol, for example, is a microtubule stabilizing drug that prevents depolymerization of the microtubules. Since blockage of spindle formation preferentially inhibits rapidly dividing cells, microtubule inhibitors have been effective agents against disorders which exhibit abnormal cell mitosis, such as cancer.
The products of secondary metabolic pathways in plants and microorganisms are a proven resource for structurally diverse and functionally unique small molecules which bind, covalently modify, or otherwise alter the function of proteins. Natural product ligands for human and pathogen proteins have revolutionized medicine in this century and have become both models and inspiration for the drug development enterprise. Cragg, J Nat Prod, 1997, 60:52-60; Shu, J Nat Prod, 1998, 61:1053-1071; Nicolaou et al. Angew Chem Int Ed, 2000, 39:44. A number of biological metabolites isolated from various sources, such as sponges, marine organisms and bacteria have been found to possess, in particular, anti-cancer activity. Fenical, “New pharmaceuticals from marine organisms,” Trends Biotech 1997, 15:339-341.
In 1991, Fenical and Clardy reported the composition and skeletal stereochemistry of two unique toxins extracted from tissues of the marine invertebrate Diazona angulata. Lindquist et al. J Am Chem Soc, 1991, 113:2303-2304. The structure of the major isolate, termed diazonamide B (1a, FIG. 1), was revealed upon X-ray diffraction measurements on a crystal of a p-bromobenzamide derivative (2, FIG. 1). These two new compounds represented a new class of halogenated, highly unsaturated cyclic peptides containing derivatives of three common amino acids, tyrosine (C1-C9), tryptophan (C18-C27) and valine (C31-35). (see 1 and 2, FIG. 1). These molecules possess an unusually rigid skeleton with little conformational freedom for the polycyclic core.
The diazonamides are a particularly complex expression of the more common polyoxazole/thiazole motif observed in peptidyl metabolites isolated from the marine environment. Belshaw et al. Science 1999, 284:486-489; Belshaw et al. Chem Biol, 1998, 5:373-84. Diazonamides comprise a complex arrangement of aromatic or heteroaromatic rings linked together as biaryls or as an intermediate quaternary center. Their synthesis requires three basic peptide modifications, 1) oxidative intramolecular coupling of aromatic side chains (forming cyclic biaryls and biaryl ethers), 2) electrophilic aromatic substitution, and 3) dehydrative cyclization to form oxazole and thiazole rings. An aromatic segment of unknown origin (C10-C17) and four proteinogenic amino acids have been incorporated into a rigid heterocyclic network which permits little extended conjugation of electron density. The fully substituted bis-oxazole (C26-C3 1), the C16-C18 biaryl linkage, and the hindered C10 quaternary center present a challenging molecule for synthesis.
In addition, diazonamide A has demonstrated potent antineoplastic activity. Lindquist et al. J Am Chem Soc, 1991, 113:2303-2304. In HCT-116 cells, a human colorectal carcinoma line, diazonamide exhibits GI50 values (50% growth inhibitory concentration) of less than 15 ng/ml.
Naturally occurring diazonamide A sent for differential cytotoxicity analysis in the NCI 60 cell mean graph screening profile (COMPARE analysis) identified a correlation with known anti-mitotic agents, such as vinblastine, paclitaxel (taxol) and vincristine. (See Table 1 below.) Hélène C. Vervoort, PhD thesis, 1999, “Novel anticancer agents from Ascidiacea,” University of California, San Diego (Scripps Institution of Oceanography). The NCI (National Cancer Institute) 60-cell line human tumor screen is a measure of the effectiveness of a compound for inhibiting or killing various human cancers. It is a set of 60 different cancer cell lines against which chemical compounds can be tested against to determine if the compound has anti-cancer activity. Each compound has an individual “fingerprint” based on effectiveness in killing each of the 60 cancer cell lines. The 50% growth inhibitory concentration (GI50), total growth inhibitory concentration (TGI), 50% lethal concentration (LC50) for any single cell line are indexes of cytotoxicity or cytostasis. A pairwise correlation coefficient (PCC) is calculated for each compound in the database. Those compounds with the highest correlation coefficient are most similar to diazonamides. As a result, these compounds represent a new class of anti-tumor agents.
TABLE 1COMPARE Analysis - Diazonamide in comparisonto known anti-mitotic compounds.GI50TGILC50Compound(PCC)Compound(PCC)Compound(PCC)Vinblastine0.696Vinblastine0.679Mitindomide0.992(antimitotic)(antimitotic)(Topo. IIInhibitor)Maytansine0.622Maytansine0.615Tetraplatin0.961(antimitotic)(antimitotic)(DNAAlkylating)Paclitaxel0.618Vincristine0.6105-FUDR0.929(antimitotic)(antimitotic)(RNA/DNAAnti-Metabolites)Vincristine0.598Rhizoxin0.593L-Alanosine0.815(antimitotic)(antimitotic)(RNA/DNAAnti-Metabolites)
Naturally occurring diazonamide must be isolated from the fractionation of tissues from the marine ascidian, Diazona angulata. 256.2 grams of lyophilized ascidian was originally used to collect 54 mg of diazonamide. Lindquist et al. J Am Chem Soc, 1991, 113:2303-2304. Because of the limited availability of the natural compound and its potent antimitotic properties, there have been substantial efforts made to synthesize diazonamides and their intermediates. Vedejs et al. Org. Lett., 2001, 3:2451-2454; Kreisberg et al., Tetrahedron Lett., 2001, 42:627-629; Wipf et al. Org. Lett., 2001, 3:1261-1264; Nicolaou et al., Angew Chem., 2000, 112:3615-3620; Fuerst et al., Org. Lett., 2000, 2:3521-3523; Bagley et al., Tetrahedron Lett., 2000, 41:6897-6900; Bagley et al., Tetrahedron Lett., 2000, 41:6901-6904; Lach et al. Tetrahedron Lett., 2000, 41:6893-6896; Vedejs et al. Org. Lett., 2000, 2:1031-1032; Vedejs et al. Org. Lett., 2000, 2:1033-1035; Magnus et al. Tetrahedron Lett., 2000, 41:831-834; Chan et al., Tetrahedron Lett., 2000, 41:835-838; Hang et al. Synthesis, 1999, 398-400; Magnus et al. Tetrahedron Lett., 1999, 40:451-454; Boto et al. Tetrahedron Lett., 1998, 39:8167-8170; Wipf et al. Tetrahedron Lett., 1998, 39:2223-2226; Jamison, T. F., PhD thesis, Harvard University, Cambridge Mass, 1997; Moody et al. J Chem Soc Perkin Trans, 1996, 16:2413-2419, Moody et al. Pure Appl Chem, 1994, 66:2107-2110; Ritter and Carreira, Angew Chem Int Ed. 2001, 41:2489-2495.
The challenge in synthesizing diazonamide arises from the core of the molecule, comprising a halogenated heterocyclic framework in a single atropisomeric form with a triaryl acetaldehyde and a quaternary C10 center (see 1 and 2, FIG. 1). Various strategies have been used to produce intermediates for diazonamide synthesis. Nicolaou and coworkers directed their effort towards the quaternary C10 center and the heterocyclic core of diazonamide. Nicolaou et al., Angew Chem., 2000, 112:3615-3620; Nicolaou et al., Angew Chem Int Ed, 2000, 39:3473-3478. They employed the Horner-Wadsworth-Emmon cyclization technique to induce ring closure of a benzofuranone-derived intermediate. They also utilized an intramolecular pinacol cyclization strategy of an aldehyde and an oxime. Nicolaou et al., Angew Chem., 2001, 113:4841-4845; Nicolaou et al., Angew Chem Int Ed 2001, 40:4705-4709. Wipf et al. discloses the approach of producing a bis-oxazoyl indole similar to that found in diazonamide A using consecutive Chang rearrangements. The endproduct of this effort does not achieve a ring closed material. Wipf et al., Org. Lett., 2001, 3:1261-1264. Vedejs and coworkers have produced a intermediate with the desired stereochemistry and ring closure using imino-Dieckman cyclization. Vedejs et al. Org. Lett., 2001, 3:2451-2454; Vedejs et al. Org. Lett., 2000, 2:1031-1032; Vedejs et al. Org. Lett., 2000, 2:1033-1035.
In an alternative approach, Magnus et al. disclosed the use of the photo-Fries rearrangement strategy to produce an intermediate that achieves ring closure and exhibits the desired stereochemistry, but does not possess the C 10 quaternary center. Magnus et al. Tetrahedron Lett., 2000, 41:831-834. The aforementioned attempts have not produced compounds with a diazonamide-like skeleton and exhibiting antimitotic activity.
The present invention describes the first successful laboratory synthesis of any diazonamide compound with antimitotic activity. Li et al. “Total synthesis of nominal diazonamides—Part I:Convergent preparation of the structure proposed for (−)-diazonamide A,” Angew Chem Int Ed, 2001, 40:4765-4769. The method described herein achieves completely synthetic diazonamide compounds through the combined use of catalytic Heck endocyclization, stereo controlled ring-contracting pinacol rearrangement, and indole arylation via internal photoinduced electron transfer.